Immunoassay techniques have been known for the last few decades and are now commonly used in medicine for a wide variety of diagnostic purposes to detect target analytes in a biological sample. Immunoassays exploit the highly specific binding of an antibody to its corresponding antigen, wherein the antigen is the target analyte. Typically, quantification of either the antibody or antigen is achieved through some form of labeling such as radio- or fluorescence-labeling. Sandwich immunoassays involve binding the target analyte in the sample to the antibody site (which is frequently bound to a solid support), binding labeled antibody to the captured analyte, and then measuring the amount of bound labeled antibody, wherein the label generates a signal proportional to the concentration of the target analyte inasmuch as labeled antibody does not bind unless the analyte is present in the sample.
Among the currently available assays for diagnosing a clinical condition, assessing risk or predicting an outcome resulting from cardiac myocyte damage are those determining the concentration of cardiac troponin-I, cardiac troponin-T, creatine phosphokinase MB (CKMB), myoglobin, myosin heavy chain, myosin light chain, B-type natriuretic peptide (including pro-BNP, NT-proBNP, and hBNP(1-32)), heart fatty-acid-binding protein (H-FABP), placenta growth factor (PLGF), and/or interleukin-6 (IL-6). Typically, such assays are conducted as a panel of immunoassays that are performed sequentially on a test sample from a patient and testing for each analyte thus requires additional increments of time, volume of sample and reagents for each assay result. Frequently, assays for the complete panel of analytes are not available on a single assay platform, from a single vendor, or even by a single detection technology, which adds to the complexity of assessing the clinical status of the patient. Further, when conducted separately and in series, the result for any single analyte in the panel may be unreliable, i.e. may or may not be erroneous. Which if any of the individual assay results are erroneous may be difficult or impractical to determine, thus leading to potentially conflicting results and correspondingly conflicting diagnosis, assessment of risk or prediction of outcome.
The example of two related antigens, cardiac troponin-T and cardiac troponin-I is illustrative. Cardiac troponin-I assays are readily commercially available from multiple vendors, but cardiac troponin-T assays are restricted to a single vendor, Roche Diagnostics, which means that no alternatives are available. Additionally, both assays are limited in terms of accuracy according to multiple reports of false-positive and false negative results. (See, e.g, P. R. Kenny et al., J. 32 Rheumatol, 1258-61, (2005); R. I. Knoblock et al., Archives of pathology & laboratory medicine 2002; 126:606-9; and A. N. Makaryus et al., Clin Cardiol 2007; 30:92-4). Moreover, while both cardiac troponin-I and cardiac troponin-T in theory are associated with cardiac myocyte damage, immunoassays for the determination of cardiac troponin-I and cardiac troponin-T in patient samples fail to correlate about 10% of the time. This discrepancy may be due to differences in antibody configurations, other biochemical differences or analytical among the different assays.
Further, such immunoassays for circulating antigens are known for being susceptible to interference from other substances that may be present in a test sample, such as heterophilic endogenous antibodies, and autoantibodies. Such interference is typically addressed by performing a second assay to identify problematic samples, which is time-consuming and costly. Another approach to addressing the autoantibody problem is to choose analyte-specific antibodies that bind to specific epitopes distinct from the analyte epitopes that react with the autoantibodies. Following this general approach, efforts have focused on exploring the use of thousands of different combinations of two, three and even four analyte-specific antibodies to avoid interference from autoantibodies. However, this effort has been largely unsuccessful. It is now evident that autoantibodies against complex protein analytes are likely to be polyclonal within a particular sample, and may be even more diverse among samples from different individuals. Interference from diverse polyclonal autoantibodies may explain the observation that as little as 25% or even less of an analyte protein sequence binds to analyte-specific antibodies, which may in turn explain the lack of success using this approach.
Thus, at least for the aforementioned reasons, initial tests of either cardiac troponin-I or cardiac troponin-T alone and using standard assays may be negative despite the occurrence of MI, i.e. may provide false negative results. While the available assays for cardiac troponin-I or cardiac troponin-T have improved the timeliness of diagnosis of MI, nevertheless many patients with MI symptoms are not positively diagnosed until several hours after presentation. This results in delayed or postponed treatment with potentially serious results.
A need exists in the art for new immunoassay methods that improve detection of cardiac myocyte damage including the timeliness of such detection, and also for methods that compensate for interference by heterophilic endogenous antibodies and autoantibodies that may be present in a test sample, and in particular, for such methods that achieve these results without redesign of the analyte detection or capture antibodies.